Stress proteins and uses therefor

ABSTRACT

The present invention relates to stress proteins and methods of modulating an individual&#39;s immune response. In particular, it relates to the use of such stress proteins in immune therapy and prophylaxis, which results in an induction or enhancement of an individual&#39;s immune response and as an immunotherapeutic agent which results in a decrease of an individual&#39;s immune response to his or her own cells. The present invention also relates to compositions comprising a stress protein joined to another component, such as a fusion protein in which a stress protein is fused to an antigen. Further, the present invention relates to a method of generating antibodies to a substance using a conjugate comprised of a stress protein joined to the substance.

RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.08/336,251, filed Nov. 3, 1994, now abandoned, which is acontinuation-in-part of the corresponding International ApplicationPCT/US94/06362, filed Jun. 6, 1994 and U.S. Ser. No. 08/073,381, filedJun. 4, 1993, now abandoned, which is a Continuation-in-Part of U.S.Ser. No. 07/804,632 filed Dec. 9, 1991, now abandoned, which is aFile-Wrapper-Continuation of U.S. application Ser. No. 07/366,581 filedJun. 15, 1989, now abandoned, which is a Continuation-in-Part of U.S.application Ser. No. 07/207,298 filed Jun. 15, 1988, now abandoned, andthe corresponding International Application PCT/US89/02619 filed Jun.15, 1989. The teachings of PCT/US94/06362, U.S. Ser. No. 08/073,381,U.S. Ser. No. 07/804,632, U.S. Ser. No. 07/366,581, U.S. Ser. No.07/207,298 and PCT/US89/02619 are incorporated herein by reference.

FUNDING

Work described herein was funded by grants from the National Institutesof Health (AI23545), the World Health Organization Program for VaccineDevelopment, and the World Health Organization/World Bank/United NationsDevelopment Program Special Program for Research and Training inTropical Diseases. The United States government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Although the function of stress proteins is not entirely clear, itappears that some participate in assembly and structural stabilizationof certain cellular and viral proteins, and their presence at highconcentrations may have an additional stabilizing effect during exposureto adverse conditions. Neidhardt, F. C. and R. A. Van Bogelen, In:Escherichia coli and Salmonella typhimurium, Cellular and MolecularBiology, (eds. Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik,B. Schaechter, M. and Umbarger, H. E. (Am. Soc. Microbiol., Washington,D.C.), pp. 1334-1345 (1987); Pelham, H. R. B. Cell, 46:959-961 (1986);Takano, T. and T. Kakefuda, Nature, 239:34-37 (1972); Georgopoulos, C.et al., New Biology, 239:38-41 (1972). Phagocytic host cells produce ahostile environment of foreign organisms, and the ability to producestress proteins has been implicated in the survival of bacterialpathogens within macrophages Christman, M. F. et al., Cell, 41:753-762(1985).

Mycobacterium (M.) tuberculosis and Mycobacterium (M.) leprae are theetiologic agents of tuberculosis and leprosy, respectively. Thesediseases afflict 20-30 million people and continue to present asignificant global health problem. Joint International Union AgainstTuberculosis and World Health organization Study Group, Tubercle,63:157-169 (1982); Bloom, B. and T. Godal, Rev. Infect Dis. 5:765-780(1983). To develop more effective tools for the diagnosis and preventionof these diseases, it is important to understand the immune response toinfection by mycobacterial pathogens.

The antibody and T-cell responses to infection or inoculation withkilled mycobacteria have been studied in humans and in animals. Humanpatients with tuberculosis or leprosy produce serum antibodies directedagainst at least 12 mycobacterial proteins. Some of these proteins arealso recognized by well-characterized murine monoclonal antibodies. Miceimmunized with mycobacterial lysates produce antibodies that aredirected predominantly to six M. tuberculosis and six M. leprae proteinantigens. Engers, H. D. Infect. Immun., 48:603-605 (1985); Engers, H.D., Infect. Immune., 51:718-720 (1986). Genes encoding these 12mycobacterial antigens have been cloned, and recombinant proteinsproduced from these clones have been used to investigate the humanT-lymphocyte response to mycobacterial infection. Husson, R. N. and R.A. Young, Proc. Natl. Acad. Sci., USA, 84:1679-1683 (1987); Young, R. A.et al., Nature, 316:450-452 (1985); Britton, W. J. et al., Lepr. Rev.,57, Suppl. 2, 67-75 (1986).

Protection against mycobacterial disease involves cell-mediatedimmunity. Joint International Union Against Tuberculosis and WorldHealth Organization Study Group, Tubercle, 63:157-169 (1982); Hahn, H.and S. H. E. Kaufman, Rev. Infect. Dis., 3:1221-1250 (1981).T-lymphocytes cloned from patients or from volunteers immunized withkilled mycobacteria have been tested for their ability to recognize therecombinant mycobacterial proteins. Lymphocyte-proliferation assaysdemonstrate that most of the antigens identified with monoclonalantibodies are involved in the T-cell response to mycobacterialinfection or vaccination in mice and in humans. Limiting dilutionanalysis indicates that 20% of the mycobacterial-reactive CD4⁺T-lymphocytes in mice immunized with M. tuberculosis recognize a singleprotein, the 65-kDa antigen. Kaufman, S. H. E. et al., Eur J. Immunol.,17:351-357 (1987).

SUMMARY OF THE INVENTION

The present invention relates to stress proteins and methods ofmodulating an individual's (such as a human, other mammal or othervertebrate) immune response. In particular, it relates to the use ofsuch stress proteins in immune therapy or prophylaxis, which results inan induction or enhancement of an individual's immune response and as animmunotherapeutic agent which results in a decrease of an individual'sresponse to his or her own cells. In the embodiment in which anindividual's immune response is induced or enhanced, the induced orenhanced response can be a response to antigens, such as those derivedfrom a pathogen or cancer cell, or can be upregulation of theindividual's immune status, such as in an immune compromised individual.In immune prophylaxis, stress proteins are administered to prevent orreduce the effects in an individual of a pathogen, which can be anyvirus, microorganism, parasite or other organism or substance (e.g., atoxin or toxoid) which causes disease or to prevent or reduce theeffects in an individual of cancer cells. In preventing or reducingadverse effects of pathogens which contain stress proteins (e.g.,bacteria, parasite, fungus) according to the method of the presentinvention, an individual's immune response to the pathogen's stressprotein(s) is induced or enhanced through the administration of avaccine which includes the pathogen's stress protein(s) or other stressproteins. The stress protein can be administered alone, as a member orcomponent of a conjugate (e.g., joined to another antigen by chemical orrecombinant means such as joined to a fusion partner resulting in afusion protein), or as an adjuvant or carrier molecule to enhance orobtain a desired immune response to an antigen.

The present invention also relates to compositions which are conjugatescomprised of a stress protein joined to another substance or component.For example, the present invention relates to a conjugate in which astress protein is chemically linked to an antigen, or in which a stressprotein is fused to an antigen (e.g., a fusion protein).

The present invention also relates to a method of generating monoclonalor polyclonal antibodies to a substance using a conjugate comprised of astress protein joined to the substance. In this embodiment, an effectiveamount of the conjugate (i.e., an amount which results in an immuneresponse in the host) is introduced into a mammalian host which resultsin production of antibodies to the substance in the host. The antibodiesare removed from the host and purified using known techniques (e.g.,chromatography).

Preventing or reducing adverse effects of viral pathogens which do or donot contain stress proteins, as well as preventing or reducing theadverse effects of cancer cells according to the present method, iseffected by enhancing an individual's immune surveillance system.Enhancement of immune response can be effected by modulating the immunecells by stimulation with a stress protein (e.g., a bacterial stressprotein).

In the embodiment in which an individual's immune response is decreased,such as is used in treating autoimmune diseases, stress proteins knownto be involved in the autoimmune response are administered to turn downan individual's immune response by tolerizing the individual to thestress proteins. Alternatively, the immune response to stress protein,which is known to occur in autoimmune disease, is reduced by interferingwith the ability of immune cells which respond to stress proteins to doso.

A selected stress protein of the present invention can be administeredto an individual, according to the method of the present invention, andresult in an immune response which provides protection againstsubsequent infection by a pathogen (e.g., bacteria, other infectiousagents which produce stress proteins) or reduction or prevention ofadverse effects of cancer cells. Alternatively, a selected stressprotein can be administered to an individual, generally over time, toinduce immune tolerance against the selected stress protein. Forexample, a selected stress protein can be administered in multiple dosesover time in order to induce immune tolerance against an autoimmunedisease such as rheumatoid arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph illustrating the sequence similarity between portionsof the M. tuberculosis 71-kDa antigen (residues 1-204; TB 71 kDa) andthe E. coli DnaK protein (residues 430-639).

FIG. 1B is a graph illustrating the sequence similarity between portionsof the M. tuberculosis 65-kDa antigen (residues 1-540; TB 65 kDa) andthe E. coli GroEL protein (residues 1-547).

FIGS. 2A-2B are a comparison of the amino acid sequence of the human P1protein (573 residues) (SEQ ID NO: 1) and the amino acid sequence of thegroEL protein (547 residues) (SEQ ID NO: 2).

FIGS. 3A-3B are a comparison of the amino acid sequence of the human P1protein (573 residues) (SEQ ID NO: 1), which is a homolog of groELprotein, and the amino acid sequence of the 65 kDa M. leprae protein(540 residues) (SEQ ID NO: 3).

FIGS. 4A-4B are a comparison of the amino acid sequence of the human P1protein (573 residues) (SEQ ID NO: 1), which is a homolog of the groELprotein, and the amino acid sequence of the 65kDa M. tuberculosisprotein (540 residues) (SEQ ID NO: 4).

FIG. 5 is a schematic representation of selected stress protein fusionvectors which contain a polylinker with multiple cloning sitespermitting incorporation of a gene of interest.

FIG. 6 is a schematic representation of the stress protein fusionvector, pKS70 containing the T7 RNA polymerase promoter, a polylinkerand the mycobacterial tuberculosis hsp70 gene, and the stress proteinfusion vector pKS72 containing the HIV p24 gag gene subcloned into thepKS70 vector.

FIG. 7 is a graph illustrating the anti-p24 antibody titer in miceinjected with the p24-hsp70 fusion protein, p24 alone and hsp70 alone.

DETAILED DESCRIPTION OF THE INVENTION

Cells respond to a variety of stressful stimuli by increasing thesynthesis of specific stress proteins. The most extensively studiedcellular response to stressful stimuli is the synthesis of heat shockproteins (hsp) by a cell, induced by a sudden increase in temperature.Because many of the heat shock proteins are also induced by otherstresses, they are frequently called stress proteins. Stress proteinsand their relatives appear to help assemble and disassemble proteincomplexes. In bacteria, the major stress proteins, hsp70 and hsp60,occur at moderate levels in cells that have not been stressed butaccumulate to very high levels in stressed cells. For example, hsp70 andhsp60 normally account for 1-3% of total E. coli protein, but canaccumulate to about 25% under stressful conditions. Eukaryotic hsp70 andhsp60 proteins do not accumulate to these extreme levels. Their levelsrange from undetectable to moderately abundant, depending on theorganism and cell type.

The present invention is based on the observation that stress proteinsare among the major antigens available for presentation to T lymphocytesand may be common immune targets in a broad spectrum of infectiousdiseases. Immune responses to stress proteins are involved in immunesurveillance by the body and a variety of different T cell types hasbeen shown to recognize highly conserved stress protein determinants.Several observations, described below, suggest a model of immunesurveillance in which self-reactive T cells provide a first line ofdefense against infection or other invasion by pathogens, which include,but are not limited to, viruses, microorganisms, other organisms,substances such as toxins and toxoids, and agents which cause celltransformation, by recognizing and helping to eliminate stressedautologous cells, as well as cells infected with intracellularpathogens. Without wishing to be bound by this model, it is presented asone means by which it is possible to explain why prokaryotic andeukaryotic cells respond to a variety of potentially damaging stimuli,such as elevated temperature, by increasing the synthesis of a family ofproteins, referred to as stress proteins, which are among the mosthighly conserved and abundant proteins found in nature.

Investigation of antigens involved in the immune response to thetuberculosis and leprosy bacilli (M. tuberculosis and M. leprae)initially led to the observation that a variety of stress proteins areamong the major targets of the immune response, as is described atgreater length below.

Further assessment has demonstrated that stress proteins may be commonimmune targets in a broad spectrum of infectious diseases. Sequenceanalysis has revealed 70-kDa heat shock protein homologues among majorantigens of the protozoan parasites Plasmodium falciparum (Bianco, A. E.et al., Proc. Natl. Acad. Sci., USA, 83:8713-8717 (1986)) andSchistosoma mansoni (Hedstrom, R. et al., J. Exp. Med., 165:1430-1435(1987)) and the malarial parasite Brugia malayi (Selkirk, M. E. et al.,J. Cell Biochem., 12D:290 (1988)). Similarly, homologues of GroEL havebeen found among antigens involved in the immune response to Salmonellatyphimurium and Coxiella (Vodkin, M. H. and J. C. Williams, J.Bacteriol, 170:1227 (1988)), as well as Bordetella pertussis (DelGiudice, G., et al., J. of Imm., 150: 2025-2032 (1993)). The presence ofstress proteins among major immune targets in a variety of humanpathogens is support for the idea that the stress response may be ageneral component of infection and that stress proteins should beconsidered among candidates for subunit vaccines. All organisms respondto heat by inducing synthesis of heat shock proteins (hsp), which are agroup of proteins. This response is the most highly conserved geneticsystem known and has been shown to occur in every organism, includingmicroorganisms, plants and animals, investigated to date. Many of thecharacteristics of the response are common to all organisms and the hspare among the most highly conserved proteins known. For example, hsp90family and hsp70 family proteins are present in widely diverseorganisms. The proteins in each family—even in such diverseorganisms—show approximately 50% identity at the amino acid level and atthe nonidentical residues, exhibit many similarities. Several of theproteins induced by heat are also induced by a variety of otherstresses. The hsps or a closely related/similar protein are present inall organisms at normal temperatures and have been shown to have keyfunctions in normal cell metabolism. Lindquist, S. and E. A. Craig, Ann.Rev. Genet., 22:631-677 (1988). Because the stress response is common toprokaryotes and eukaryotes and stress proteins are among the most highlyconserved in sequence, it is reasonable to expect that an antigen fromone pathogen could immunize against another pathogen. Exposure toforeign stress proteins early in life might, in fact, induce a degree ofimmunity to a variety of infectious agents. If so, this could provide anexplanation for the observation that, for many pathogens, only afraction of infected individuals actually acquire clinical disease.

The following is a description of the relationship which has beenobserved between stress proteins and the immune response tomycobacterial infection; of the observation and supporting informationthat stress proteins are immune targets in many infections by pathogens;of the role of stress proteins as immune targets in transformed cells;of recognition of the fact that the immune response to conserved stressprotein determinants may play an important role in autoimmune pathologyin rheumatoid arthritis, as well as in adjuvant arthritis; and of therole of stress proteins in immune surveillance, as well as a modelproposed for immune surveillance in which self-reactive T cells providea first line of defense against infection and cell transformation.

Mycobacterial Stress Proteins are Targets of the Immune Response

An intriguing relationship between stress proteins and the immuneresponse to mycobacterial infection has been observed. A more detailedexamination of stress protein determinants and immune responsemechanisms is essential to understanding the relationship among stressproteins, infection, and immunity.

In view of the involvement of proteins of M. tuberculosis and M. lepraein humoral and cell-mediated immune responses and to establish thefunctions of these proteins in the mycobacterial cell, the DNA encodingseveral of the M. tuberculosis and M. leprae antigens have beensequenced. The results, discussed in Example 1, demonstrate that many ofthese mycobacterial protein antigens exhibit striking sequencesimilarity to known stress-induced proteins. Three of the M. leprae andtwo of the M. tuberculosis protein antigens studied have been shown toexhibit striking sequence similarity to known stress proteins. Forreasons discussed in Example 1, it is concluded that two of the M.leprae and two of the M. tuberculosis antigens are homologues of the E.coli DnaK and GroEL proteins.

In mice, immunization with mycobacterial lysates elicits antibodyresponses to at least six M. tuberculosis protein antigens and a similarnumber of M. leprae protein antigens. Monoclonal antibodies specific forthese proteins have been used to isolate clones from λgtll DNAexpression libraries of M. tuberculosis and M. leprae. The sequence ofthe DNA clones revealed that mycobacterial hsp70 (alias 70 kDa antigen)and hsp60 (alias 65 kDa antigen, GroEL) were the major targets of themurine antibody response to both M. tuberculosis and M. leprae. Twoadditional hsp, an 18 kDa member of the small hsp family and a 12 kDahomologue of groES, were found among the M. leprae and M. tuberculosisantigens. Young, D. B., et al., Proc. Natl. Acad. Sci., USA,85:4267-4270 (1988); Shinnick, T. M., et al., Nuc. Acids Res., 17:1254(1989).

The mycobacterial stress proteins are among the immunodominant targetsof both murine antibody and T cell responses. In one study whichsummarized results obtained from 10 laboratories, a collection of 24murine monoclonal antibodies recognized 6 M. leprae proteins; 7 of theseantibodies are directed against 6 different determinants in the M.leprae hsp60. Engers, H. D., et al., Infect. Immune., 48:603-605 (1985);Mehra, V., et al., Proc. Natl. Acad. Sci., USA, 83:7013-7017 (1986). Ina similar study, 3 of 33 monoclonal antibodies raised against M.tuberculosis recognized the M. tuberculosis hsp60 protein. Engers, H.D., et al., Infect. Immune., 1:718-720 (1986). Finally, limitingdilution analysis indicates that 20% of the mycobacterial-reactive CD4+T lymphocytes in mice immunized with M. tuberculosis recognize thisantigen. Kaufmann, S. H., et al., Eur. J. Immunol., 17:351-357 (1987).

Although a rigorous quantitative analysis of the human immune responseto mycobacterial stress proteins has not yet been reported,mycobacterial stress proteins are recognized by human antibodies and Tlymphocytes and the evidence suggests that these proteins are among themajor targets of the human cell mediated immune response. Emmrich. F.,et al., J. Exp. Med., 163:1024-1029 (1985);. Mustafa, A. S., et al.,Nature (London). 319:63-66 (1986); Oftung, F., et al., J. Immunol.,138:927-931 (1987); Lamb, J. R., et al., EMBO J., 6:1245-1249 (1987). Tlymphocytes from patients with mycobacterial infection or fromvolunteers immunized with mycobacteria have been cloned and tested fortheir ability to recognize the mycobacterial stress proteins. In each ofthese studies, some fraction of the human T cell clones were shown torecognize one or more of the mycobacterial stress proteins.

Stress Proteins are Immune Targets in Infections by Pathogens

The observation that stress proteins are important targets of the immuneresponse to mycobacterial infection and the knowledge that the majorstress proteins are conserved and abundant in other organisms suggestedthat stress proteins are likely to be immune targets in many infectionsby pathogens. Indeed, that is now clearly the case. Antigens from a widevariety of infectious agents have been identified as members of stressprotein families. The major stress protein antigen recognized byantibodies in bacterial infections is hsp60. “Common antigen”, animmunodominant protein antigen long known to be shared by most bacterialspecies, turns out to be hsp60. Shinnick, T. M., et al., Infect.Immune., 56:446 (1988); Thole, J. E. R., et al., Microbial Pathogenesis,4:71-83 (1988). Stress proteins have also been identified as immunetargets in most major human parasite infections. Bianco, A. E., et al.,Proc. Natl. Acad. Sci. USA, 83:8713 (1986); Nene, V., et al., Mol.Biochem. Parasitol., 21:179 (1986); Ardeshir, F., et al., EMBO J., 6:493(1987); Hedstrom, R., et al., J. Exp. Med., 165:1430 (1987); Selkirk, M.E., et al., J. Cell Biochem., 12D:290 (1988), Engman, D. M., et al., J.Cell Biochem., 12D: Supplement, 290 (1988); Smith, D. F., et al., J.Cell Biochem., 12D:296 (1988). Antibodies to hsp70 have been identifiedin the sera of patients suffering from malaria, trypanosomiasis,leishmaniasis, schistosomiasis and filariasis. Hsp90 is also a target ofantibodies in trypanosomiasis and a member of the small hsp family isrecognized in some patients with schistosomiasis.

Proteins homologous to stress proteins have also been identified inviruses. Recently, a protein encoded by the RNA genome of the BeetYellows Closterovirus, a plant virus, has been shown to be homologous tohsp70. Agranovsky, A. A., et al., J. Mol. Biol., 217: 603-610 (1991). Inaddition, stress protein induction occurs in eukaryotic cells followinginfection by diverse viruses in vitro. Collins, P. L., and Hightower, L.E., J. Virol., 44:703-707 (1982); Nevins, J. R., Cell, 29:913-939(1982); Garry, R. F. et al., Virology, 129:391-332 (1988); Khandjian, E.W. and Turler, H., Mol. Cell Biol., 3:1-8 (1983); LaThangue, N. B., etal., EMBO J., 3:267-277 (1984); Jindal, S. and Young, R., J. Viral,66:5357-5362 (1992). CTL that recognize these neo-antigens could limitthe spread of virus by killing infected cells, possibly beforesubstantial amounts of mature virus are assembled, and by secreting thelymphokine γ-interferon. Pestka, S., in: Methods Enzymol., Interferons,Part A., Vol. 79 Academic Press, New York, pp. 667 (1981). Evidenceconsistent with this idea is emerging. Koga et al., (1989) have shownthat infection of primary murine macrophages with CMV rendered themsusceptible as targets for MHC-I restricted CD8⁺ CTL specific for linearepitopes of M. tuberculosis hsp60. Koga, T., et al. (1989). Although theepitope recognized by these CTL on infected macrophages was not defined,it is tempting to speculate that a cross-reactivity with self hsp60epitopes is being observed. Indeed, the same groups showed that ahomologous hsp60 is constitutively present in macrophages and isupregulated by γ-interferon stimulation.

Stress Proteins as Immune Targets in Transformed Cells

Stress proteins appear to be produced at high levels in at least sometransformed cells. Bensaude, O. and Morange, M., EMBO J., 2: 173-177(1983). An 86 kDA murine tumor antigen has been found to be homologousto representatives of the hsp90 family in yeast and Drosophila. Ullrich,S. J., Proc. Natl. Acad. Sci., USA, 83: 3121-3125 (1986). Immunizationof mice with the purified protein led to inhibition of tumor growth in95% of experimental animals that had been seeded with cultured tumorcells. All of the protected mice had high titers of anti-hsp90 serumantibody which was able to precipitate murine hsp90 from lysates of heatshocked mouse embryo cells. Again, a role for autoreactive lymphocytesis implied, since T cells capable of recognizing autologous cellsstressed by transformation could help eliminate nascent tumor cells.

Stress Proteins and Autoimmune Processes

Rheumatoid arthritis is characterized by a chronic proliferative andinflammatory reaction in synovial membranes which is thought to involveautoimmune processes. Rat adjuvant arthritis resembles human rheumatoidarthritis in many respects, and has been used as an experimental animalmodel for human disease. Pearson, C. M., Arthritis Rheum., 7:80-86(1964). Adjuvant arthritis can be induced in rats with a singleintradermal injection of killed M. tuberculosis in complete Freund'sadjuvant. An autoimmune process involving T lymphocytes appears to beresponsible for the generation of the disease. Holoshitz, J., et al.,Science, 219:56-58 (1983). T cell lines isolated from the draining lymphnodes of arthritic rats and propagated in vitro by stimulation with M.tuberculosis-pulsed syngeneic antigen presenting cells can cause atransient form of the disease when transferred to irradiated rats. Sincecare was taken in these experiments to exclude the transfer ofcontaminating M. tuberculosis , this result strongly suggests that theclinical effects of the disease are a consequence of an autoimmunereaction in which the autoantigen is shared with M. tuberculosis.

The rat and M. tuberculosis antigens recognized by the arthritogenic Tcells have been sought for a number of years. A number of differentproteins present in synovial membranes have been proposed to be thecross-reactive rat antigen, but were later discounted as procedures forthe purification of these proteins improved. van Eden, W., et al., Proc.Natl. Acad. Sci., USA, 82:5117-5120 (1985); Holoshitz, J., et al.,Science, 219:56-58 (1983). The M. tuberculosis antigen recognized by thearthritogenic T cells was recently shown to be a 65 kDa protein (vanEden, W., et al., Nature, 331:171 (1988), which has now been shown to behsp60 (see the Example 1). Using a combination of truncated recombinant65 kDa proteins and peptides, a nine amino acid epitope of hsp60 hasbeen identified as the minimum stimulatory sequence for arthritogenic Tcell clones in proliferation assays. Now that it is clear that somearthritogenic T cells recognize the mycobacterial hsp60, it is quitepossible that the rat autoantigen is also hsp60.

The results obtained in the adjuvant arthritis model led investigatorsto determine whether T lymphocytes from human rheumatoid arthritispatients also recognize mycobacterial antigens. These investigators havefound not only that patients with rheumatoid arthritis have T cells thatrecognize M. tuberculosis antigens, but that these T cells have diversephenotypes. Substantial proliferative responses to mycobacterialextracts are observed with uncloned T cells (predominantly CD4⁺) fromboth synovial infiltrates and peripheral blood, although responses aregenerally greater in synovial infiltrates. Abrahamson, T. G., et al.,Scand. J. Immunol., 7:81-90 (1978); Holoshitz, J., et al., Lancet ii,305-306 (1986). Holoshitz et al. found that 4 of 5 T cell clonesisolated from human rheumatoid synovia which respond to M. tuberculosisantigens were CD4⁻ CD8⁻ cells with γ/δ T cell receptors. Holoshitz, J.,et al., Nature, 339:226-229 (1989). This observation is interestingbecause γ/δ T cells have yet to be assigned a role in immunity. One ofthe γ/δ clones was tested for its ability to respond to purifiedmycobacterial hsp60 and was found to be positive in proliferationassays. Due to the conserved nature of stress proteins, these T cellshave the potential for autoreactivity. Lamb and coworkers have shownthat polyclonal T cells from synovial infiltrates recognize bothmycobacterial hsp60 and hsp70 . Lamb, J. R., et al., Intl. Immunol., inpress (1989). The population of T cells that recognize the mycobacterialstress proteins were shown to respond to E. coli hsp60 and hsp70 and,most interestingly, human hsp70 purified from heat shocked macrophages.Thus, immune responses to conserved stress protein determinants, perhapsinitiated by bacterial infection (not necessarily by mycobacteria), mayplay an important role in autoimmune pathology in rheumatoid arthritis,as well as in adjuvant arthritis.

Stress Proteins and Immune Surveillance

A variety of different T cell types has now been shown to recognizehighly conserved stress protein determinants. The ability of cells torespond to stress by increasing the levels of the highly conservedstress proteins; the presence of T cells of diverse phenotypes inhealthy individuals that are capable of recognizing self stress proteindeterminants; and observations that stress responses are induced bypathogenic infections and by cell transformation, all suggest a model ofimmune surveillance in which self-reactive T cells provide a first lineof defense against infection and transformation by recognizing andhelping to eliminate stressed autologous cells, as well as cellsinfected with intracellular pathogens. The pool of lymphocytes thatrecognize conserved stress protein determinants might be induced duringestablishment of natural microbial flora on the skin and in the gut, andmaintained by frequent stimulation by pathogens, such as bacteria andviruses, as well as other stressful stimuli encountered during a normallifetime. This model is attractive because it provides a way in whichthe immune system could exploit the existence of conserved epitopes instress proteins to respond immediately to antigenically diversepathogens and cellular changes, producing an initial defense that neednot await the development of immunity to novel antigens.

The lymphocytes which recognize conserved stress protein determinantsmust be capable of discriminating between normal and stressed cells.Since many stress proteins are constitutively expressed in normal cells,although at lower levels than in stressed cells, the potential forautoreactivity is ever-present. Normal cells may escape destruction byexpressing only substimulatory levels of stress protein determinants ontheir surfaces. In addition, stress proteins may only be processed andpresented during stress, and it may be relevant that many stressproteins have altered intracellular locations during stress. Finally,immune regulatory networks may prevent activation of autoreactive Tcells under normal conditions. The regulatory constraints required bythis system might occasionally break down, perhaps during stress causedby bacterial or viral infections, leading to autoimmune disease.Rheumatoid arthritis may be such a disease.

Modulation of Immune Response

The precise relationship between stress proteins and the host immuneresponse to infection is as yet undefined. When cells are subjected to avariety of stresses, they respond by selectively increasing thesynthesis of a limited set of stress proteins. Some stress proteins,including the products of DnaK and GroEL, are major constituents of thecell under normal growth conditions and are induced to even higherlevels during stress. Lindquist, S., Annu. Rev. Biochem. 55: 1151-1191(1986); Neidhardt, F. C. and R. A. VanBogelen, In Escherichia coli andSalmonella TVphimurium, Cellular and Molecular Biology, (eds. Neidhardt,F. C., Ingraham, J. L. Low, K. B. Magasanik, B. Schaechter, M. andUmbarger, H. E.) Am. Soc. Microbiol., Washington, D.C., pp. 1134-1345(1987). It has now been demonstrated that stress-related proteins aretargets of the immune response. Young, D. et al., Proc. Natl. Acad. Sci.USA, 85:4267-4270 (1988). It is reasonable to expect that immunodominantantigens would be found among such abundant proteins, as has now beenshown to be the case.

According to the method of the present invention, it is possible tomodulate the immune response in an individual, such as a human, othermammal or other vertebrate, by altering the individual's response tostress proteins. In particular, it is possible to enhance or induce anindividual's response to a pathogen (e.g., bacteria, virus, parasites,or other organism or agent, such as toxins, toxoids) or to cancer cellsor enhance or induce an upregulation of an individual's immune status(such as in an immune compromised individual or HIV-infectedindividual); and to decrease an individual's autoimmune response, suchas occurs in some forms of arthritis. In addition, administration of astress protein using the method of the present invention providesprotection against subsequent infection by a pathogen. As demonstratedherein, stress proteins contain regions of highly conserved amino acidsequences and have been shown to be major immunodominant antigens inbacterial and other infections. Therefore, it is reasonable to expectstress proteins can be used to elicit strong immune responses against avariety of pathogens. The stress protein administered to induce orenhance an immune response to pathogens can be the stress protein of thepathogen against which an immune response is desired or other stressprotein, a portion of that protein of sufficient size to stimulate thedesired immune response or a protein or amino acid sequence which is thefunctional equivalent of the stress protein in that it is sufficientlyhomologous in amino acid sequence to that of the stress protein to becapable of eliciting the desired response (an immune responsesubstantially similar to that which occurs in response to the stressprotein) in the individual to whom it is administered. The term“sufficiently homologous in amino acid sequence to that of the stressprotein” means that the amino acid sequence of the protein orpolypeptide will generally show at least 40% identity with the stressprotein amino acid sequence; in some cases, the amino acid sequence of afunctional equivalent exhibits approximately 50% identity with the aminoacid sequence of the stress protein.

Any stress-induced proteins or their functional equivalents can be usedby the present invention to enhance or induce an immune response in anindividual (e.g. a human, other mammal or vertebrate), against aninfection by a pathogen, for immunotherapy against cancer cells, forgenerally upregulating an individual's immune status and for use ininducing immune tolerance in an individual or animal.

The stress proteins of the present invention can be administered in avariety of ways to modulate the immune response of an individual (e.g.,a human, other mammal or other vertebrate). In one embodiment, thestress protein is administered as a vaccine which is comprised of thestress protein or a portion of the stress protein which is of sufficientsize to stimulate the desired immune response. In this embodiment, thevaccine can be a “specific vaccine” which contains a specific stressprotein of a particular pathogen against which an immune response isdesired, such as a bacterial stress protein. In this case, since thepathogen's stress proteins are distinguishable from those of the host,it is possible to induce an immunoprophylactic response specific to thepathogen's stress proteins. Blander, S. J., et al., J. Clin. Invest.,91:717-723 (1993). This can be carried out by administering a vaccinewhich includes all or a portion (e.g., sufficient amino acid sequence tohave the desired stimulatory effect on immune response) of thepathogen's stress protein or of another protein having an amino acidsequence sufficiently similar to that of the stress protein sequence tostimulate the immune response to the pathogen's stress protein.Alternatively, in the case of a pathogen which does not contain stressproteins, (e.g. some viruses) or in the condition of neoplasia, stressproteins or highly conserved stress protein determinants, such as thoseshown to be recognized by a variety of T cells, can be administered as atype of “general” vaccine to achieve an upregulation of the immuneresponse. Administration of such a vaccine will enhance the existingimmune surveillance system. For instance, a vaccine which includes abacterial, or other stress protein can be administered to enhance theimmune system which will result in an immune response against a pathogenwhich does not contain stress proteins. Alternatively, this type of“general” vaccine can be used to enhance an individual's immune responseagainst cancer or to generally upregulate an individual's immune status,such as in an immune compromised individual (e.g., an individualundergoing chemotherapy or an HIV-infected individual). In either caseof this embodiment (specific or general vaccine), the immune response tothe stress protein sequence will be increased and effects of thepathogen, disease condition or immune impairment will be reduced(decreased, prevented or eliminated).

In another embodiment, stress proteins can be used to enhance immunesurveillance by applying local heat or any other substances or changesin condition which induce the stress response in the individual beingtreated. (This can also be employed in conjunction with the specificvaccine, described previously, administered to enhance an immuneresponse to a stress protein-containing pathogen or in conjunction withthe general vaccine, described above, administered to enhance the immuneresponse against a pathogen which does not contain its own stressproteins, cancer, or to upregulate the immune status of an individual).For example, it is known that increased levels of stress proteins areproduced in many types of cancer cells. Therefore, enhancement of theimmune surveillance system, using this embodiment of the presentinvention as described, can be used to facilitate destruction and/or toprevent progression or establishment of cancer cells.

The method of the present invention can also be used to modify ormodulate an individual's response to his or her own cells (e.g., as inautoimmune diseases). There are at least two ways in which the presentinvention can be used immunotherapeutically. First, stress proteins,such as heat shock proteins (e.g., hsp 70 and hsp60 ), are known to beinvolved in autoimmune disease. It is, thus, possible to turn down anindividual's immune response, resulting in the individual becoming moretolerant of the protein. Second, because it is known that under somecircumstances, one component of the immune response in certainautoimmune diseases can be to stress proteins, it is possible toselectively inhibit or interfere with the ability of immune cells whichnormally interact with such proteins to do so. This can be done, forexample, by administering monoclonal antibodies that bind to specific Tcell receptors and delete or disable such cells. Alternatively, ratherthan knocking out immune cells, the stress response in cells can beturned down by administering a drug capable of reducing a cell's abilityto undergo the stress response. For example, a drug targeted to orspecific for heat shock transcription factor, which is needed tostimulate heat shock genes, can be administered. The transcriptionfactor is rendered nonfunctional or subfunctional and, as a result,cells'ability to undergo the stress response is also lessened.

In another embodiment of the present invention, the stress protein isadministered as a vaccine which is comprised of two moieties: a stressprotein and another substance (referred to as an antigen, e.g. protein,peptide, carbohydrate, lipid, organic molecule) against which an immuneresponse is desired. The two moieties are conjugated or joined to form asingle unit. Conjugation can be achieved by chemical means known tothose skilled in the art (e.g. through a covalent bond between thestress protein and the second moiety; reductive amination) or, asdemonstrated in Example 2, by recombinant techniques. If recombinanttechniques are used to produce the conjugate, the result is arecombinant fusion protein which includes the stress protein and theantigen in a single molecule. This makes it possible to produce andpurify a single recombinant molecule in the vaccine production process.In this embodiment, the stress protein can be seen to act as anadjuvant-free carrier, and it stimulates strong humoral and T cellresponses to the substance to which the stress protein is fused. Thestress protein can be conjugated to any substance against which animmune response is desired or to a portion of the substance sufficientto induce an immune response in an individual to whom it isadministered. The substance includes but is not limited to proteins(e.g., ovalbumin, Influenza virus Hemagglutinin, Human ImmunodeficiencyVirus p24), peptides (e.g., Human Immunodeficiency Virus peptides,melanoma antigen peptides), oligosaccharides (e.g., Neiserriameningitidis group B, Streptococcus pneumoniae type 14, Hemophilisinfluenzae type b), lipids, carbohydrates (e.g., glycolipid antigens inhuman cancers such as GD3, GM2, Gb3, Forssman antigen, Sialosyl-Le^(a)antigen and glycoprotein antigens in human cancers such as CEA, AFP,PSA, Tn antigen), organic molecules or a combination thereof. Recentevidence demonstrating the effectiveness of such a vaccine indicatesthat mycobacterial hsp70 proteins when conjugated to other proteins actas adjuvant-free carriers. The humoral immune response to some peptidesconjugated to mycobacterial hsp70 administered without any adjuvant wasvery similar to the antibody response to the same peptides administeredin Freund's complete adjuvant. Lussow, A. R., et al., Eur. J. Immune.,21:2297-2302 (1991). Barrios, C. et al., Eur. J. Immune., 22:1365-1372(1992).

The present: invention also relates to compositions which are conjugatescomprised a stress protein joined to another substance or component. Forexample, the present invention relates to a conjugate in which a stressprotein is chemically linked to an antigen, or in which a stress proteinis fused to an antigen (e.g., a fusion protein).

As demonstrated in Example 3, the HIV p24 gag gene was subcloned intothe stress protein fusion vector pKS70 (FIG. 6), containing the T7 RNApolymerase promoter, a polylinker and the mycobacterial tuberculosishsp70 gene. The resulting vector pKS72 (FIG. 6) was used to produce thep24-hsp70 fusion protein in E. coli. Adjuvant-free, purified p24-hsp70fusion protein was injected into Balb/c mice and as shown in FIG. 7, theanti-p24 antibody titer was 2.7 orders of magnitude higher in miceinjected with the p24-hsp70 fusion protein than in mice injected withp24 alone or hsp70 alone. Mice injected with p24 and the adjuvant, alum,also produced an antibody response to p24. Finally, a demonstrable Tcell response was seen in mice injected with the p24-hsp70 fusionprotein and in mice injected with p24 alone.

In another embodiment of the present invention, the stress protein or aportion of the stress protein which is of sufficient size to stimulatean immune response or an equivalent, is administered as an adjuvant,with another substance (referred to as an antigen) against which animmune response is desired. The stress protein can be used as anadjuvant with any substance or antigen against which an immune responseis desired or to a portion of the substance sufficient to induce animmune response in an individual to whom it is administered. Thesubstance includes proteins, peptides, oligosaccharides, lipids,carbohydrates, organic molecules or a combination thereof. Via linkageto a stress protein, strong and specific B and T cell mediated immunitycan be generated in a mammalian host (e.g., mice, rabbits, humans) tovirtually any organic molecule. This is particularly useful 1) withsubstances (e.g., antigens) which alone are non-immunogenic; 2) whenadjuvants cannot be used or do not work well in combination with aparticular antigen; 3) when the availability of purified antigen islimited, particularly with fusion proteins where the antigen is madeusing recombinant DNA technology; 4) where other carrier molecules, suchas KLH, BSA, OVA or thyrogloulin, which additionally require adjuvants,are not effective or desirable; 5) there is a genetic restriction in theimmune response to the antigen; 6) there is a pre-existingimmunosuppression or non-responsiveness to an antigen (e.g., pediatricvaccines where infants and children under 2 years of age do not generateprotective immunity to carbohydrate antigens well); and 7) the type ofimmune response achieved by other carriers or adjuvants is undesirableor ineffectual (i.e., stress protein conjugates could be used to biastoward either B or T cell immunity via proper dose, route andinoculation regimen).

The present invention also relates to a method of generating monoclonalor polyclonal antibodies to a substance using a conjugate comprised of astress protein joined to the substance. In this embodiment, an effectiveamount of the conjugate (i.e., an amount which results in an immuneresponse in the host) is introduced into a mammalian host which resultsin production of antibodies to the substance in the host. The antibodiesare remvoed from the host and purified using known techniques (e.g.,chromatography), thereby resulting in production of polyclonalantibodies. Alternatively, the antibodies produced using the method ofthe present invetion can be used to generate hybridoma cells whichproduce monoclonal antibodies using known techniques (Kohler, G., etal., Nature, 256:495(1975) Milstein et al., Nature, 266:550-552(1977);Koprowski et al., Proc. Natl. Acad. Sci, 74:2985-2988 (1977); Welsh,Nature, 266:495(1977); Maniatis, T. et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.(1982)).

The stress protein, stress protein portion, stress protein functionalequivalent and the substance to which the stress protein is fused orconjugated present in the vaccine can be produced or obtained usingknown techniques. For example, the stress protein or stress proteinportion can be obtained (isolated) from a source in which it occurs innature, can be produced by cloning and expressing a gene encoding thedesired stress protein or stress protein portion or can be synthesizedchemically or mechanically.

An effective dosage of the stress proteins of the present invention asvaccines or adjuvants, to elicit specific cellular and humoral immunityto stress proteins, or to substances conjugated to the stress proteins,such as proteins or oligosaccharides, is in the range of 0.1 to 1000 ughsp per injection, depending on the individual to whom the stressprotein is being administered. Lussow, A. R., et al., Eur. J. Immune.,21:2297-2302 (1991). Barrios, C. et al., Eur. J. Immune., 22:1365-1372(1992). The appropriate dosage of the stress protein for each individualwill be determined by taking into consideration, for example, theparticular stress protein being administered, the type of individual towhom the stress protein is being administered, the age and size of theindividual, the condition being treated or prevented and the severity ofthe condition. Those skilled in the art will be able to determine usingno more than routine experimentation, the appropriate dosage toadminister to an individual.

Various delivery systems can be used to administer an effective dose ofthe vaccine of the present invention. Methods of introduction include,for example, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural and oral routes. Any other convenientroute of administration can be used (infusion of a bolus injection,infusion of multiple injections over time, absorption through epithelialor mucocutaneous linings such as, oral mucosa, rectal and intestinalmucosa) or a series of injections over time.

The present invention is further illustrated by the followingexemplification, which is not intended to be limiting in any way.

EXEMPLIFICATION EXAMPLE 1

Isolation and Characterization of Mycobacterial Stress Protein Antigens

Recombinant DNA Clones. The isolation and characterization of M.tuberculosis and M. leprae λgtll genomic DNA clones with murinemonoclonal antibodies have been described. Husson, R. N. and Young, R.A., Proc. Natl. Acad. Sci., USA 84: 1679-1683 (1987); Young, R. A., etal., Nature (London) 316: 450-452 (1985). DNA was isolated from theseclones and was manipulated by standard procedures. Davis, R. W.,Advanced Bacterial Genetics: A Manual for Genetic Engineering (ColdSpring Harbor Lab., Cold Spring Harbor, N.Y.), (1980).

DNA Sequence Analysis. DNA was subcloned into vector M13mp18 or M13mp19(New England Biolabs), as suggested by the supplier. Dideoxynucleotidechain-termination reactions and gel electrophoresis of the sequencedproduced were as described. Davis, R. W., Advanced Bacterial Genetics: AManual for Genetic Engineering (Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.), (1980). DNA sequences were determined for both strands ofDNA. Computer analysis of sequences with UWGCG programs was as describedby Devereux, J., et al., Nucleic Acids Res., 12: 387-395 (1984).

Immunoblot Analysis. Escherichia coli strain TG1 was transformed withthe following plasmids by standard procedures (Maniatis, T., et al.,Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, N.Y.) (1982), with selection for ampicillin resistance:pND5, a derivative of pBR325 containing the E. coli GroEL genes(Jenkins, A. J., et al., Mol. Gen. Genet., 202: 446-454 (1986); pUC8(Vic, J., Gene, 19: 259-268 (1982); pUC8 with insert DNA for λgtll cloneY3178 (M. leprae 65-kDa antigen, Young, R. A., et al., Nature, (London)316: 450-452 (1985)) ligated in the EcoRI site.

Overnight cultures of E. coli strains in Luria-Bertani (LB) medium werecentrifuged and resuspended in isotonic phosphate-buffered saline at acell density corresponding to an absorbance of 2 at 600 nm. An equalvolume of sample buffer containing 2% (wt/vol) NaDodSo₄ was added, and,after heating on a boiling water bath for 2 min, samples wereelectrophoresed on 12% (wt/vol) polyacrylamide gels in the presence ofNaDodSO₄. Blots were prepared by electrophoretic transfer of theproteins to a nitrocellulose membrane, and binding of monoclonalantibodies was assayed with a peroxidase-conjugated secondary antibodyas described. Young, D. B., et al., Infect. Immune., 55: 1421-1425(1987).

Six M. tuberculosis and six M. leprae proteins have been implicated inthe immune response to the mycobacterial pathogens (Table 1). To obtainclues to the normal cellular function of several of these mycobacterialantigens, DNA clones encoding these proteins, isolated by usingmonoclonal antibodies to probe lambda gtll libraries (Husson, R. N. andYoung, R. A., Proc. Natl. Acad. Sci., USA, 84: 1679-1683 (1987); Young,R. A., et al., Nature, (London) 316: 450-452 (1985)) were subjected tosequence analysis. The sequences elucidated have been submitted to theGenBank sequence database.

The Mycobacterial 71-k Da Antigen. The 71-k Da antigen of M.tuberculosis is recognized by human T cells during infection (Table 1).

TABLE 1 MYCOBACTERIAL PROTEIN ANTIGENS Subjected to Homology Recognizedby sequence with known Protein, kDA Human T Cells analysis proteins M.tuberculosis 71 + + DnaK 65* + + GrOEL 38 + − − 19 + + None 14 + − − 12ND − − M. leprae 70 ND − DnaK 65 + + GroEL 36 + − − 28 + − − 18 + +Plant Hsp 12 ND − −

Mycobacterial protein antigens, their recognition by human T cells, andhomology of the deduced mycobacterial protien sequences to knownproteins are summarized. ND, not determined; +, yes; −, no

Includes data derived from study of the 65-kDA antigens of M. bovis BCG(Bacillus Calmette-Gurein), which is identical to the M. tuberculosis65-kDA antigen.

A. S. Mustafa, J. R. Lamb, D. Young and R. A. Young, unpublished data.

The insert DNA of lambdagtll clone Y3271 (Husson, R. N., et al, Proc.Natl. Acad. Sci, USA, 84: 1679-1683 (1987), was sequenced to obtainamino acid sequence information for the 71-kDa antigen of M.tuberculosis. This clone produces a beta-galactosidase fusion proteincontaining the carboxyl-terminal one-third of the 71-kDa antigenexhibiting 40% amino acid sequence identity with the comparable segmentof the dnaK gene product from E. coli (Bardwell, J. C., et al., Proc.Natl. Sci., USA, 81: 848-852 (1984)), (FIG. 1). FIG. 1A shows the extentof sequence similarity between portions of the mycobacterial and the E.coli 70-k Da polypeptides. Sequences transcriptionally downstream fromthe mycobacterial 71-k Da gene predict a 356-amino acid proteinhomologous to the E. coli dnaJ gene product (unpublished data),indicating that the E. coli dnaK-dnaj operon structure is conserved inM. tuberculosis and consistent with the conclusion that themycobacterial 71-kDa antigen is a homologue of the E. coli dnaK geneproduct. The product of the dnaK gene is a member of the 70-kDa heatshock protein family that is highly conserved among prokaryotes andeukaryotes (Bardwell, J. C., et al., Proc. Natl. Acad. Sci., USA, 81:848-852 (1984); Lindquist, S., Annu. Rev. Biochem., 55: 1151-1191(1986).

The M. leprae 70-k Da antigen cross-reacts with monoclonal antibodiesdirected to the M. tuberculosis 70-kDa antigen. M. tuberculosis and M.leprae are both members of the 70-k Da heat shock protein family ofstress proteins.

The mvcobacterial 65-kDa antigen. The 65-kDa antigens of M. tuberculosisand M. leprae are involved in the human T-cell response to mycobacterialinfection (Table 1). Genes encoding these proteins have been isolated(Husson, R. N., and Young, R. A., Proc. Natl. Acad. Sci., USA, 84:1679-1683 (1987); Young, R. A., et al., Nature, (London) 316: 450-452(1985)) and sequenced (Shinnick, T. M., J. Bacteriol., 169: 1080-1088(1987); Mehram, V., et al., Proc. Natl. Acad. Sci., USA 83: 7013-7017(1986)), revealing that the amino acid sequences of the 65-kDa antigensof M. tuberculosis (SEQ ID NO: 4) and M. leprae (SEQ ID NO: 3) are 95%identical. These proteins sequences exhibited no significant sequencesimilarity to proteins in the GenBank database.

Identification of these proteins was based on the observation that somemonoclonal antibodies directed against the mycobacterial 65-kDa antigenscross-react with an E. coli protein of 60 kDa. E. coli cells transformedwith the plasmid pND5 (Sanger, F., et al., Proc. Natl. Acad. Sci., USA74: 5463-5467 (1977), which contains the E. coli gro E genes, had beenshown to accumulate large amounts of the 60-kDa protein. A comparison ofthe mycobacterial 65-kDa protein sequences with those determined for E.coli groEl (C. Woolford, K. Tilly, C. Georgopoulous, and R. H.,unpublished data) revealed the extent of the sequence similarity asshown in FIG. 1B.

The 60-kDa Gro EL protein is a major stress protein in E. coli.Lindquist, S., Annual. Rev. Biochem., 55: 1151-1191 (1986); Nature, 333:330-334 (1988). There is some evidence that the mycobacterial 65-kDaproteins accumulate in response to stress: Mycobacterium bovis BCG(bacillus Calmette-Guerin) cultures grown in zinc-deficient medium aresubstantially enriched in this protein (De Bruyn, J., et al., Infect.Immune. 55: 245-252 (1987)). This infers that the 65-kDa proteins of M.tuberculosis and M. leprae are homologues of the E. coli Gro EL protein.

Other Mycobacterial Antigens. T lymphocytes that respond to the M.tuberculosis 19-kDa antigen and the M. leprae 18-kDa antigen have beenobserved in humans with tuberculosis and leprosy, respectively (Table1). DNA encoding these antigens was sequenced from the λgtll clonesY3148 (Husson, R. N. and Young, R. A., Proc. Natl. Acad. Sci., USA 84:1679-1683 (1987); and Y3179 (Young, R. A., et al., Nature, (London) 316:450-452 (1985)), respectively. The M. tuberculosis 19-kDa proteinsequence predicted from the DNA exhibited no significant sequencesimilarity to proteins in the GenBank database.

However, the M. leprae 18-kDa protein sequence was similar to thesoybean 17-kDa protein heat shock protein, a protein representation of amajor class of plant heat. shock proteins (Schoffl, F. and Van Bogelen,R. A., In: Escherichia coli and Salmonella typhimurium, Cellular andMolecular Biology, Am. Soc. Microbiol., Washington, D.C. (1987).

EXAMPLE 2

Construction of Stress Protein-Fusion Vaccines for Use as Adiuvant-FreeCarriers in Immunizations

Recombinant Fusion Vectors. A series of stress protein fusion vectorsfor use in E. coli were constructed and are shown in FIG. 5. Thesevectors contain the T7 RNA polymerase promoter fused to the M. bovis BCGhsp70 gene or the M. bovis BCG hsp60 gene. The vectors also contain apolylinker with multiple cloning sites, permitting incorporation of agene of interest so that the antigen encoded by that gene is expressedas a fusion protein with the stress protein. A subset of these vectorspermit incorporation of the foreign gene with a coding sequence for aC-terminal 6-Histidine “tag” for ease of fusion protein purification.Thus far, recombinant clones have been generated that produce hsp70proteins fused to HIV gag and HIV pol proteins.

Purification of stress protein fusions. Two strategies have beendeveloped to purify the recombinant fusion proteins. The T7 systemusually produces such large amounts of protein that it forms inclusionbodies, permitting purification by centrifugation. The preliminaryresults indicate that an hsp70-HIV gag fusion protein accounts for about20% of total E. coli protein in the T7 system. If necessary, otherfusion proteins can be purified via the 6-Histidine “tag”.

EXAMPLE 3

Adjuvant-Free Carrier Effect of HSP70 IN Vivo

The stress protein fusion vector pKS70 (FIG. 6), containing the T7 RNApolymerase promoter, a polylinker and the mycobacterial tuberculosishsp70 gene, was constructed. The HIV p24 gag gene was subcloned intopKS70 using the Ndel and BamHI sites and the resulting pKS72 vector(FIG. 6) was used to produce the p24-hsp70 fusion protein in E. coli.The fusion protein was purified as inclusion bodies and further purifiedusing ATP-agarose chromatography and MonoQ ion exchange chromatography.

The p24-hsp70 protein in phosphate buffered saline (PBS), in the absenceof an adjuvant, was injected intraperitoneally into Balb/c mice. Ascontrols, the p24 protein alone in PBS or the hsp70 protein alone in PBSwas injected into different groups of mice. Three weeks later, the micewere boosted and finally, three weeks after the boost, the mice werebled. The anti-p24 antibody titer was then determined by ELISA. Miceinjected with 25 pmoles of p24-hsp70 had antibody levels 2.7 orders ofmagnitude higher than mice injected with p24 alone or hsp70 alone (FIG.7). Results of experiments in which mice were injected with p24 and theadjuvant, alum, also showed that there was an antibody response to p24.In addition, mice injected with the p24-hsp70 fusion protein and miceinjected with p24 alone produced a demonstrable T cell response.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

4 573 amino acids amino acid linear protein 1 Met Leu Arg Leu Pro ThrVal Phe Arg Gln Met Arg Pro Val Ser Arg 1 5 10 15 Val Leu Ala Pro HisLeu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30 Gly Ala Asp Ala ArgAla Leu Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45 Asp Ala Val Ala ValThr Met Gly Pro Lys Gly Arg Thr Val Ile Ile 50 55 60 Glu Gln Ser Trp GlySer Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80 Ala Lys Ser IleAsp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90 95 Leu Val Gln AspVal Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110 Thr Thr ThrAla Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125 Glu LysIle Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135 140 MetLeu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys 145 150 155160 Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala 165170 175 Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala Met Lys Lys180 185 190 Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly Lys Thr LeuAsn 195 200 205 Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe Asp Arg GlyTyr Ile 210 215 220 Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly Gln Lys CysGlu Phe Gln 225 230 235 240 Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys IleSer Ser Ile Gln Ser 245 250 255 Ile Val Pro Ala Leu Glu Ile Ala Asn AlaHis Arg Lys Pro Leu Val 260 265 270 Ile Ile Ala Glu Asp Val Asp Gly GluAla Leu Ser Thr Leu Val Leu 275 280 285 Asn Arg Leu Lys Val Gly Leu GlnVal Val Ala Val Lys Ala Pro Gly 290 295 300 Phe Gly Asp Asn Arg Lys AsnGln Leu Lys Asp Met Ala Ile Ala Thr 305 310 315 320 Gly Gly Ala Val PheGly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335 Val Gln Pro HisAsp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys 340 345 350 Asp Asp AlaMet Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365 Lys ArgIle Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375 380 TyrGlu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395400 Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405410 415 Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val420 425 430 Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg CysIle 435 440 445 Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gln LysIle Gly 450 455 460 Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile Pro Ala MetThr Ile Ala 465 470 475 480 Lys Asn Ala Gly Val Glu Gly Ser Leu Ile ValGlu Lys Ile Met Gln 485 490 495 Ser Ser Ser Glu Val Gly Tyr Asp Ala MetAla Gly Asp Phe Val Asn 500 505 510 Met Val Glu Lys Gly Ile Ile Asp ProThr Lys Val Val Arg Thr Ala 515 520 525 Leu Leu Asp Ala Ala Gly Val AlaSer Leu Leu Thr Thr Ala Glu Val 530 535 540 Val Val Thr Glu Ile Pro LysGlu Glu Lys Asp Pro Gly Met Gly Ala 545 550 555 560 Met Gly Gly Met GlyGly Gly Met Gly Gly Gly Met Phe 565 570 547 amino acids amino acidlinear protein 2 Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg ValLys Met 1 5 10 15 Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys ValThr Leu Gly 20 25 30 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe GlyAla Pro Thr 35 40 45 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile GluPro Glu Asp 50 55 60 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu ValAla Ser Lys 65 70 75 80 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr AlaThr Val Leu Ala 85 90 95 Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val AlaAla Gly Met Asn 100 105 110 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys AlaVal Thr Ala Ala Val 115 120 125 Glu Glu Leu Lys Ala Leu Ser Val Pro CysSer Asp Ser Lys Ala Ile 130 135 140 Ala Gln Val Gly Thr Ile Ser Ala AsnSer Asp Glu Thr Val Gly Lys 145 150 155 160 Leu Ile Ala Glu Ala Met AspLys Val Gly Lys Glu Gly Val Ile Thr 165 170 175 Val Glu Asp Gly Thr GlyLeu Gln Asp Glu Leu Asp Val Val Glu Gly 180 185 190 Met Gln Phe Asp ArgGly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro 195 200 205 Glu Thr Gly AlaVal Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp 210 215 220 Lys Lys IleSer Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val 225 230 235 240 AlaLys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly 245 250 255Glu Ala Leu Ala Thr Ala Val Val Asn Thr Ile Arg Gly Ile Val Lys 260 265270 Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275280 285 Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu290 295 300 Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly GlnAla 305 310 315 320 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile IleAsp Gly Val 325 330 335 Gly Glu Glu Ala Ala Ile Gln Gly Arg Val Ala GlnIle Arg Gln Gln 340 345 350 Ile Glu Glu Ala Thr Ser Asp Tyr Asp Arg GluLys Leu Gln Glu Arg 355 360 365 Val Ala Lys Leu Ala Gly Gly Val Ala ValIle Lys Val Gly Ala Ala 370 375 380 Thr Glu Val Glu Met Lys Glu Lys LysAla Arg Val Glu Asp Ala Leu 385 390 395 400 His Ala Thr Arg Ala Ala ValGlu Glu Gly Val Val Ala Gly Gly Gly 405 410 415 Val Ala Leu Ile Arg ValAla Ser Lys Leu Ala Asp Leu Arg Gly Gln 420 425 430 Asn Glu Asp Gln AsnVal Val Ser Ser Ser Leu Arg Ala Met Glu Ala 435 440 445 Pro Leu Arg GlnIle Val Leu Asn Cys Gly Glu Glu Pro Ser Val Val 450 455 460 Ala Asn ThrVal Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala Ala 465 470 475 480 ThrGlu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro Thr 485 490 495Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly Leu 500 505510 Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp Ala 515520 525 Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met Gly530 535 540 Gly Met Met 545 540 amino acids amino acid linear protein 3Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu 1 5 1015 Arg Gly Leu Asn Ser Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro 20 2530 Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile 35 4045 Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro 50 5560 Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr 65 7075 80 Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln 8590 95 Ala Leu Val Lys Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro100 105 110 Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Asp Lys Val ThrGlu 115 120 125 Thr Leu Leu Lys Asp Ala Lys Glu Val Glu Thr Lys Glu GlnIle Ala 130 135 140 Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile GlyAsp Leu Ile 145 150 155 160 Ala Glu Ala Met Asp Lys Val Gly Asn Glu GlyVal Ile Thr Val Glu 165 170 175 Glu Ser Asn Thr Phe Gly Leu Gln Leu GluLeu Thr Glu Gly Met Arg 180 185 190 Phe Asp Lys Gly Tyr Ile Ser Gly TyrPhe Val Thr Asp Ala Glu Arg 195 200 205 Gln Glu Ala Val Leu Glu Glu ProTyr Ile Leu Leu Val Ser Ser Lys 210 215 220 Val Ser Thr Val Lys Asp LeuLeu Pro Leu Leu Glu Lys Val Ile Gln 225 230 235 240 Ala Gly Lys Ser LeuLeu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala 245 250 255 Leu Ser Thr LeuVal Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val 260 265 270 Ala Val LysAla Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln 275 280 285 Asp MetAla Ile Leu Thr Gly Ala Gln Val Ile Ser Glu Glu Val Gly 290 295 300 LeuThr Leu Glu Asn Thr Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys 305 310 315320 Val Val Met Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp 325330 335 Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Thr Glu Ile Glu340 345 350 Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg LeuAla 355 360 365 Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala AlaThr Glu 370 375 380 Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp AlaVal Arg Asn 385 390 395 400 Ala Lys Ala Ala Val Glu Glu Gly Ile Val AlaGly Gly Gly Val Thr 405 410 415 Leu Leu Gln Ala Ala Pro Ala Leu Asp LysLeu Lys Leu Thr Gly Asp 420 425 430 Glu Ala Thr Gly Ala Asn Ile Val LysVal Ala Leu Glu Ala Pro Leu 435 440 445 Lys Gln Ile Ala Phe Asn Ser GlyMet Glu Pro Gly Val Val Ala Glu 450 455 460 Lys Val Arg Asn Leu Ser ValGly His Gly Leu Asn Ala Ala Thr Gly 465 470 475 480 Glu Tyr Glu Asp LeuLeu Lys Ala Gly Val Ala Asp Pro Val Lys Val 485 490 495 Thr Arg Ser AlaLeu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Thr 500 505 510 Thr Glu AlaVal Val Ala Asp Lys Pro Glu Lys Thr Ala Ala Pro Ala 515 520 525 Ser AspPro Thr Gly Gly Met Gly Gly Met Asp Phe 530 535 540 540 amino acidsamino acid linear protein 4 Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu AlaArg Arg Gly Leu Glu 1 5 10 15 Arg Gly Leu Asn Ala Leu Ala Asp Ala ValLys Val Thr Leu Gly Pro 20 25 30 Lys Gly Arg Asn Val Val Leu Glu Lys LysTrp Gly Ala Pro Thr Ile 35 40 45 Thr Asn Asp Gly Val Ser Ile Ala Lys GluIle Glu Leu Glu Asp Pro 50 55 60 Tyr Glu Lys Ile Gly Ala Glu Leu Val LysGlu Val Ala Lys Lys Thr 65 70 75 80 Asp Asp Val Ala Gly Asp Gly Thr ThrThr Ala Thr Val Leu Ala Gln 85 90 95 Ala Leu Arg Lys Glu Gly Leu Arg AsnVal Ala Ala Gly Ala Asn Pro 100 105 110 Leu Gly Leu Lys Arg Gly Ile GluLys Ala Val Glu Lys Val Thr Glu 115 120 125 Thr Leu Leu Lys Gly Ala LysGlu Val Glu Thr Lys Glu Gln Ile Ala 130 135 140 Ala Thr Ala Ala Ile SerAla Gly Asp Gln Ser Ile Gly Asp Leu Ile 145 150 155 160 Ala Glu Ala MetAsp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu 165 170 175 Glu Ser AsnThr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg 180 185 190 Phe AspLys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg 195 200 205 GlnGlu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys 210 215 220Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly 225 230235 240 Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala245 250 255 Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys SerVal 260 265 270 Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala MetLeu Gln 275 280 285 Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser GluGlu Val Gly 290 295 300 Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu GlyLys Ala Arg Lys 305 310 315 320 Val Val Val Thr Lys Asp Glu Thr Thr IleVal Glu Gly Ala Gly Asp 325 330 335 Thr Asp Ala Ile Ala Gly Arg Val AlaGln Ile Arg Gln Glu Ile Glu 340 345 350 Asn Ser Asp Ser Asp Tyr Asp ArgGlu Lys Leu Gln Glu Arg Leu Ala 355 360 365 Lys Leu Ala Gly Gly Val AlaVal Ile Lys Ala Gly Ala Ala Thr Glu 370 375 380 Val Glu Leu Lys Glu ArgLys His Arg Ile Glu Asp Ala Val Arg Asn 385 390 395 400 Ala Lys Ala AlaVal Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr 405 410 415 Leu Leu GlnAla Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp 420 425 430 Glu AlaThr Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu 435 440 445 LysGln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu 450 455 460Lys Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly 465 470475 480 Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val485 490 495 Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu PheLeu 500 505 510 Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu LysAla Ser 515 520 525 Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe 530535 540

We claim:
 1. A method of inducing or enhancing an immune response in apatient, the method comprising administering to the patient apharmaceutical composition comprising an isolated fusion proteincomprising a stress protein joined via a peptide bond to a heterologousprotein or peptide wherein the fusion protein, when administered to thepatient, induces or enhances an immune response against the heterologousprotein or peptide.
 2. The method of claim 1, wherein the stress proteinis a mycobacterial stress protein.
 3. The method of claim 1, wherein thestress protein is a member of one of the following families of stressproteins: the hsp70 family, the hsp60 family; The groES family; the DnaJfamily; the hsp90 family; and the small molecular weight family ofstress proteins.
 4. The method of claim 1, wherein the stress protein isan M. bovis BCG hsp65 protein.
 5. The method of claim 1, wherein thefusion protein consists of a stress protein joined via a peptide bond tothe hererologous protein or peptide.
 6. A method of inducing orenhancing an immune response in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising anisolated fusion protein comprising a stress protein joined via a peptidebond to a viral antigen, wherein the fusion protein, when administeredto the patient, induces or enhances an immune response against the viralantigen.
 7. The method of claim 6, wherein the stress protein is amycobacterial stress protein.
 8. The method of claim 6, wherein thestress protein is a member of one of the following families of stressproteins: the hsp70 family; the hsp60 family; the groES family; The DnaJfamily; the hsp90 family; and the small molecular weight family ofstress proteins.
 9. The method of claim 6, wherein the stress protein isan M. bovis BCG hsp65 protein.
 10. The method of claim 6, wherein thestress protein is a heat shock protein (hsp) aid the viral antigen is ahuman immunodeficiency virus (HIV) protein or peptide.
 11. The method ofclaim 10, wherein the HIV protein or peptide is a gag or pol protein orpeptide.
 12. The method of claim 10, wherein the HIV protein or peptideis a p24 protein or peptide.
 13. The method of claim 6, wherein thefusion protein consists of a stress protein joined via a peptide bond toa viral antigen.
 14. A method of inducing or enhancing an immuneresponse in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising an isolated fusionprotein comprising a stress protein joined via a peptide bond to acancer antigen, wherein the fusion protein, when administered to thepatient, induces or enhances an immune response against the cancerantigen.
 15. The method of claim 14, wherein the stress protein is amycobacterial stress protein.
 16. The method of claim 14, wherein thestress protein is a member of one of the following families of stressproteins: the hsp70 family; the hsp60 family; the groES family; the DnaJfamily; the hsp90 family; and the small molecular weight family ofstress proteins.
 17. The method of claim 14, wherein the stress proteinis an M. bovis BCG hsp65 protein.
 18. The method of claim 14, whereinthe fusion protein consists of a stress protein joined via a peptidebond to a cancer antigen.
 19. A method of inducing or enhancing animmune response in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising an isolated fusionprotein comprising a portion of a stress protein joined via a peptidebond to a heterologous protein or peptide, wherein the fusion protein,when administered to the patient, induces or enhances an immune responseagainst the heterologous protein or peptide.
 20. The method of claim 19,wherein the stress protein is a mycobacterial stress protein.
 21. Themethod of claim 19, wherein the stress protein is a member of one of thefollowing families of stress proteins: the hsp70 family; the hsp60family; the groES family; the DnaJ family; the hsp90 family; and thesmall molecular weight family of stress proteins.
 22. The method ofclaim 19, wherein the stress protein is an M. bovis BCG stress protein.23. The method of claim 19, wherein the fusion protein consists of aportion of a stress protein joined via a peptide bond to a heterologousprotein or peptide.
 24. A method of inducing or enhancing an immuneresponse in a patient, the method comprising administering to thepatient a pharmaceutical composition comprising an isolated fusionprotein comprising a portion of a stress protein joined via a peptidebond to a viral antigen, wherein the fusion protein, when administeredto the patient, induces or enhances an immune response against the viralantigen.
 25. The method of claim 24, wherein the stress protein is amycobacterial stress protein.
 26. The method of claim 24, wherein thestress protein is a member of one of the following families of stressproteins: the hsp70 family; the hsp60 family; the groES family; the DnaJfamily; the hsp90 family; and the small molecular weight family ofstress proteins.
 27. The method of claim 24, wherein the stress proteinis an M. bovis BCG hsp65 protein.
 28. The method of claim 24, whereinthe stress protein is a heat shock protein (hsp) and the viral antigenis a human immunodeficiency virus (HIV) protein or peptide.
 29. Themethod of claim 28, wherein the HIV protein or peptide is a gag or polprotein or peptide.
 30. The method of claim 28, wherein the HIV proteinor peptide is a p24 protein or peptide.
 31. The method of claim 24,wherein the fusion protein consists of a portion of a stress proteinjoined via a peptide bond to a viral antigen.
 32. A method of inducingor enhancing an immune response in a patient, the method comprisingadministering to the patient a pharmaceutical composition comprising anisolated fusion protein comprising a portion of a stress protein joinedvia a peptide bond to a cancer antigen, wherein the fusion protein, whenadministered to the patient, induces or enhances an immune responseagainst the cancer antigen.
 33. The method of claim 32, wherein thestress protein is a mycobacterial stress protein.
 34. The method ofclaim 32, wherein the stress protein is a member of one of the followingfamilies of stress proteins: the hsp70 family; the hsp60 family; thegroES family; the DnaJ family; the hsp90 family; and the small molecularweight family of stress proteins.
 35. The method of claim 32, whereinthe stress protein is an M. bovis BCG hsp65 protein.
 36. The method ofclaim 32, wherein the fusion protein consists of a portion of a stressprotein joined via a peptide bond to a cancer antigen.